Methods of inhibiting helicobacter pylori

ABSTRACT

This invention relates to methods of screening molecules capable of inhibiting the survival of  Helicobacter pylori  in vivo by specifically inhibiting the activity of UreI, to the molecules identified by these methods, and to the use of these molecules to treat or prevent  H. pylori  infection.

[0001] This invention relates to methods of screening molecules capableof inhibiting the survival of Helicobacter, particularly Helicobacterpylori, in vivo by specifically, inhibiting the activity of UreI, to themolecules identified by these methods, and to the use of these moleculesto treat or prevent Helicobacter infection.

BACKGROUND OF INVENTION

[0002]Helicobacter priori is a microaerophilic Gram-negative bacterium,which colonizes the gastric mucosa of humans (10). H. pylori isassociated with gastritis and peptic ulcer disease and has been shown toincrease the risk of gastric cancers. Urease is a major virulence factorof H. pylori. It is involved in neutralizing the acidic microenvironmentof the bacterium and also plays a role in H. pylori metabolism (11, 26).

[0003] The urease region of the H. pylori genome is composed of two geneclusters common to all strains (9 and FIG. 1), one comprising the ureABgenes encoding the structural urease subunits and the other containingthe ureEFGH genes encoding the accessory proteins required for nickelincorporation into the urease active site. The UreI gene liesimmediately upstream from this latter gene cluster and is transcribed inthe same direction (FIG. 1). The ureA, ureB, ureE, ureF, ureG, ureH, andureI genes and gene products have been described and claimed in U.S.Pat. No. 5.695,931 and allowed patent application Ser. No. 08/472,285.both of which are specifically incorporated herein by reference.

[0004] The distances separating UreI from ureE (one base pair, bp) andureE from ureF ( 11 bp) suggest that ureI-ureE-ureF constitute anoperon. Cotranscription of ureI and ureE has been demonstrated bynorthern blot analysis (1). An H. pylori N6 mutant with a ureI genedisrupted by a MiniTn3-Km transposon was previously described by Ferreroet al. (1994) (13). This strain (N6-ureI::TnKm-8) presented a ureasenegative phenotype, so it was concluded that ureI was an accessory generequired for full urease activity.

[0005] The sequences of UreI from H. pylori and the AmiS proteins,encoded by the aliphatic amidase operons of Pseudomonas aeruginosa andRhodococcus sp. R312, are similar (5, 27). Aliphatic amidases catalyzethe intracellular hydrolysis of short-chain aliphatic amides to producethe corresponding organic acid and ammonia. It has been shown that H.pylori also has such an aliphatic amidase, which hydrolyzes acetamideand propionamide in vitro (23).

[0006] In view of the sequence similarity between UreI and AmiS togetherwith the very similar structures of the urease and amidase substrates(urea: NH₂—CO—NH₂ and acetamide: CH₃—CO—NH₂) and the production ofammonia by both enzymes, a better understanding of the function of theH. pylori UreI protein is required. This understanding will open newopportunities for the prevention and treatment of H. pylori infections.

SUMMARY OF THE INVENTION

[0007] This invention provides methods for identifying molecules capableof inhibiting the growth and/or survival of Helicobacter species,particularly, H. pylori, in vivo. In particular, the methods of thisinvention involve screening molecules that specifically inhibit UreIprotein function.

[0008] The invention encompasses the molecules identified by the methodsof this invention and the use of the molecules by the methods of thisinvention to treat or prevent Helicobacter, and particularly H. pylori,infection in humans and animals.

[0009] Another aspect of this invention is a method of preventing ortreating Helicobacter species infection by administration to a human oranimal in need of such treatment a molecule capable of inhibiting thegrowth and/or survival of Helicobacter species in vivo. One suchmolecule according to the invention is characterized by a high affinityfor UreI, which allows it (i) to be transported inside the Helicobactercell, or (ii) to inhibit transport properties of UreI, or (iii) toinhibit UreI function by inhibiting UreI interaction with urease orother Helicobacter proteins. By inhibiting UreI, such molecule rendersthe bacteria more sensitive to acidity.

[0010] Yet another aspect of this invention is the production ofimmunogenic UreI antigens and their use as vaccines to preventHelicobacter species infection and/or colonization of the stomach or thegut. Antibodies to these UreI antigens are also encompassed within thescope of this invention.

[0011] This invention further relates to recombinant strains of H.pylori comprising a modified ureI gene, such that the products of themodified gene contribute to the attenuation of the bacteria's ability tosurvive in vivo and thus, its pathogenic effects.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1 depicts the urease gene cluster of H. pylori parentalstrains N6 and SS1 and of the derived mutants deficient in UreI, strainsN6-823, N6-834, and SS1-834. The genes are indicated by boxes Kith anarrow showing the direction of their transcription. The distancesbetween the ure genes are given in base pairs, bp. The site hybridizingto the primers used to confirm correct allelic exchange in strainsN6-823, N6-834, and SS1-834 is shown. Blank boxes represent thecassettes containing the genes conferring resistance to Cm (cat) or toKm (aphA-3). The urease activity of these strains is given on theright-hand side of the figure. Urease activity was measured as therelease of ammonia on crude extracts of bacteria grown 48 hours on bloodagar plates as described previously (9). One unit corresponds to theamount of enzyme required to hydrolyze 1 μmol of urea min⁻¹ mg⁻¹ totalprotein. The data are means±standard deviation calculated from 3 to 5determinations.

[0013]FIG. 2A depicts a restriction map of pILL823, pILL824, pILL833 andpILL834. Small boxes mark the vector of each plasmid, and large boxescorrespond to genes. Ori indicates the position of the ColE1 origin ofreplication. SPR and ApR are the genes conferring resistance tospectinomycin and ampicillin, respectively. Cassettes inserted into ureIand conferring resistance to chloramphenicol (cat) or kanamycin (aphA-3)are also shown. The sequence of the DNA region comprising the ureI stopcodon and the ureE start codon, including the BclI site where adaptorH19 was inserted, is given. Insertion of H19 into the BclI site ofpILL824 produced pILL825, the resulting ureI-ureE intergenic region isalso shown. The stop codon of ureI and the start codon of ureE are boxedand the ribosome binding site (RBS) is underlined. Brackets indicate theposition of restriction sites removed by ligation.

[0014]FIG. 2B depicts a restriction map of two H. pylori/E. coli shuttleplasmids: pILL845 and pILL850. Small boxes mark the vector of eachplasmid, and large boxes correspond to genes. Ori indicates the positionof the E. coli ColE1 origin of replication and repA the gene coding forthe RepA protein necessary for autonomous replication of the pHe12 in H.pylori. Cm^(P) a marks the gene conferring resistance tochloramphenicol. The ureI promoter is represented by a “P” with an arrowindicating the direction of the transcription. The other symbols are asin FIG. 1.

[0015]FIG. 3 shows the alignment of the amino acid sequence of UreI fromH. pylori with those of similar proteins and prediction of thetwo-dimensional structure of members of the UreI/AmiS protein family.Residues identical at one position in, at least, four sequences areboxed, and dashes indicate gaps inserted to optimize alignment. Theorganisms from which the sequences originated and the degree of identitywith the H. pylori UreI protein are: UreI-Hp, Helicobacter pylori (195residues, accession No. M84338); UreI-Hf. Helicobacter felts (74%identity over 196 residues, accession No. A41012); UreI-Lacto,Lactobacillus fermentum (55% identity over the 46 residues-long partialsequence, accession No. D10605); UreI-Strepto. Streptococcus salivarius(54% identity over the 129 residues-long partial sequence, accession No.U:35248); AmiS-Myco, Mycobacterium smegmatis (39% identity over 172residues, accession No. X57175); AmiS-Rhod, Rhodococcus sp. R312 (37%identity over 172 residues accession No. Z46523) and AmiS-Pseudo,Pseudomonas aeruginosa (37% identity over 171 residues, accession No.X77161). Predicted transmembrane ∀-helices are shown as shaded boxes.The regions separating these boxes are hydrophilic loops labeled “IN”when predicted to be intracellular and “OUT” when predicted to beextracellular.

[0016]FIG. 4 depicts the kinetics of ammonium release by the N6 parentalstrain (panel A) and the UreI-deficient strain N6-834 (panel B).Bacteria (2×10⁸/ml) were harvested and washed (as described inSkouloubris et al. (30)) resuspended in 10 ml of phosphate saline buffer(PBS) at pH 7.5 to 2.2 in the presence of 10 mM urea. After 0, 3, 5 and30 minutes, 0.5 ml were withdrawn and centrifuged to eliminate bacteria.The supernatant was kept on ice until ammonium concentration wasmeasured using the assay commercialized by Sigma (kit reference #171).

[0017] Table 2 shows the results obtained with the in vitro viabilitytests and the pH measurements.

[0018] Table 3 gives the values of ammonium production by strain N6 andN6-834 presented on the graphs of FIG. 4.

DETAILED DESCRIPTION

[0019] The urease cluster of Helicobacter species is unique among themany urease operons of Gram-negative bacteria that have been sequenced(20) in that it has an extra gene, UreI. The function of UreI hastherefore been the subject of much speculation. It has mostly beenattributed the function of an accessory protein required for nickelincorporation at the urease active site or a nickel transporter. A H.pylori strain carrying a deletion of ureI replaced by a non-polarcassette (Kanamycin resistance cassette) has been constructed and wasnamed N6-834 (30). The strain has been deposited at C.N.C.M. (CollectionNationale de Culture de Microorganismes, 25 rue du Docteur Roux, 75724Paris Cédex 15, France) on Jun. 28, 1999. This is the first time that anon-polar cassette (19) has been shown to be functional in H. pylori.These results provide a valuable tool for genetic analysis of complex H.pylori operons, such as Cag. a multigenic pathogenicity island.

[0020] Studies with this strain demonstrated that UreI is not requiredfor full activity of H. pylori urease as measured after in vitro growthat neutral pH. This result argues against UreI being involved in nickeltransport since such a protein, NixA (3) already identified in H.pylori, is necessary for full urease activity. Comparing ureasesexpressed from a UreI-deficient strain and the corresponding parentalstrain show that (i) they present the same activity optimum pH (pH 8);(ii) the urease structural subunits, UreA-B, are produced in equalamounts; and (iii) the urease cellular location is identical.

[0021] It is demonstrated here that (i) UreI is essential forcolonization of mice by H. pylori; (ii) UreI is important for survivalof H. pylori at acidic pH; and (iii) UreI is necessary for urease“activation” at low pH.

[0022]H. pylori during the colonization process of the stomach has todeal with important pH variations and especially has to adapt rapidly toextremely acidic pH (as acidic as pH 1.4). We have shown that UreI isrequired for H. pylori adaptation to acidity, consistently with theabsence of colonization of the mouse stomach. As an essential proteinfor the H. pylori resistance to acidity, UreI certainly plays a key rolein the infection, establishment, and persistence of H. pylori. UreI hasa sequence similar to those of the AmiS proteins, proposed to beinvolved in the transport of short-chain amides (27), moleculesstructurally similar to urea. The UreI/AmiS proteins have thecharacteristics of integral membrane proteins, probably of thecytoplasmic membrane.

[0023] Different roles for UreI can be proposed. For instance, UreImight be involved in transport (import or export) of urea or short chainamides specifically active at low pH. However, an essential role forUreI as an amide transporter is less likely because a SS1 mutant,deficient in aliphatic amidase, colonizes as efficiently as the parentalstrain in mouse colonization experiments. In addition, amidase activityis not significantly modified by the deletion of ureI in the N6-834mutant strain (C.N.C.M. filed on Jun. 28,1999). Import or export of ureacould be consistent with the existence of a urea cycle, which is one ofthe characteristics of H. pylori (28).

[0024] Alternatively, UreI might be involved in an active ammoniumexport system. Finally, UreI might be involved in a mechanism ofcoupling urease activity to the periplasmic pH, allowing urease tobecome more active when extracellular pH is acidic.

[0025] Our results are compatible with the first hypothesis of UreIbeing an urea transporter active at acidic pH values and the thirdhypothesis of UreI being a kind of sensor protein between theperiplasmic pH and urease activity. We think that these two hypothesisare not exclusive. Whatever the role of UreI, as a membrane proteinessential for the survival of H. pylori in vivo, it now provides apowerful target for a new eradication therapy and for vaccines againstH. pylori.

[0026] Molecules capable of inhibiting the growth and/or survival ofHelicobacter in vivo may be identified by contacting a parentalHelicobacter strain with said molecule in a biological sample; testingand comparing, in the presence or absence of urea, the sensitivity tothe extracellular pH of the parental strain to a strain deficient inUreI and to a UreI deficient strain complemented with ureI selectingsaid molecules displaying a differential effect on the parental orcomplemented strain as compared to the UreI deficient strain; andcollecting said active molecule.

[0027] A molecule active specifically on UreI will be the one renderingH. pylori sensitive to acidic pH (pH 2.2) in the presence of ureawithout affecting the strain behavior at neural pH. Sensitivity toacidity in the presence of urea can be tested on whole H. pylori cellsfollowing a protocol described in the examples and adapted from Clyne etal. (8). We are now trying to transpose this test in E. coli whole cellscarrying the complete urease gene cluster on a plasmid (ureAB-ureIEFGH).Screening for a molecule rendering this recombinant E. coli moresensitive to acidity in the presence of urea will be performed asdescribed for H. pylori in the examples. To distinguish betweeninhibitory molecules acting on UreI and those acting on urease, themedium pH after whole cell incubation at pH 7 in the presence of ureawill be measured. Interesting molecules are those affecting response toacidity without inhibiting the alkalization of the medium observed afterincubation at neutral pH.

[0028] These methods may be used to identify molecules that inhibit anyHelicobacter species carrying a UreI-homolog. This includes the gastricHelicobacter species: Helicobacter pylori, Helicobacter felis,Helicobacter mustelae, Helicobacter muridarum, and also Helicobacterheilmannii, Helicobacter canis, Helicobacter bilis, Helicobacterhepaticus, and Helicobacter troguntum.

[0029] The molecules identified by the methods of this invention will becapable of inhibiting UreI activity by (i) inhibiting transport of ureaor short chain amides, (ii) inhibiting ammonium export, or (iii)inhibiting urease “activation” at low pH. The molecules according topoint (i) and (ii) should be able to diffuse throughout the outermembrane and should be active even at low concentration. Suitablecandidate molecules are structural analogs of urea or short chainamides, ammonium derivatives or urease inhibitors. For example,molecules derived from AHA (acetohydroxamic acid), hydroxyurea, hippuricacid, flurofamide, hydroxylamine, methylurea, thiourea (29), ormethylammonium. The molecules according to point (iii) should inhibitthe contact between UreI (probably inserted in the cytoplasmic membrane)and periplasmic, membrane, or cytoplasmic H. pylori proteins, which arenecessary for urease “activation” at low pH. These proteins could be thestructural subunits of urease itself, the accessory proteins, or otherproteins. Molecules obtained according to this invention should not beurease competitive inhibitors, should not be toxic or mutagenic in vivoand could potentialize the action of antibiotics or bactericidalmolecules. Validation of the action of such molecules could be performedin vivo in the mouse animal model with the pair of isogenic strains SS1and SS1-834 as described in the examples.

[0030] One example of a molecule according to this invention is amonoclonal or polyclonal antibody specific for UreI. Preferably, theantibody is capable of specifically inhibiting UreI activity.

[0031] The molecules of this invention may be administered incombination with a pharmaceutically acceptable carrier to a patientsuffering from a Helicobacter infection. Alternatively, immunogeniccompositions comprising one or more molecules according to thisinvention may be administered in a vaccine composition to preventinfection by Helicobacter species.

[0032] Immunogenic compositions according to this invention may alsocomprise all or part of the UreI protein. Preferably, the UreI fragmentscomprise at least 10 consecutive amino acids of the native UreI sequenceand more preferably, the fragments comprise at least 18, 20, or 25consecutive amino acids of the native UreI sequence. Other suitable UreIfragments may contain at least 40 or at least 100 consecutive aminoacids of the native UreI sequence. Suitable fragments of Helicobacterpylori include, for example, fragments selected from the groupconsisting of amino acid residues 22 to 31, 49 to 74, 94 to 104, and 123to 142 of H. pylori (GenBank accession No. M84338)

[0033] Reference will now be made to the following Examples. TheExamples are purely exemplary of the invention and are not to beconstrued as limiting of the invention.

EXAMPLES

[0034] Construction of defined mutations of the H. pylori ureI gene

[0035]H. pylori strains with defined mutations in ureI were generated byallelic exchange to determine whether the UreI protein was necessary forproduction of active urease. For this purpose, two plasmids (pILL823 andpILL834) with cassettes carrying antibiotic resistance genes inserted inureI were constructed in E. coli.

[0036] In one plasmid. pILL823 (FIG. 2A), the ure] gene was inactivatedby insertion of a promoterless cat gene. conferring resistance tochloramphenicol (Cm). A 780 bp blunt-ended BamHI restriction fragmentcontaining the “cat cartridge” from pCM4 (Pharmacia, Sweden) wasintroduced into a unique HpaI site, between codons 21 and 22 of ureI, inpILL753 (9). In the resulting plasmid, pILL823 (FIG. 2A), cat is in thesame orientation as ureI and is expressed under the control of the ureIpromoter.

[0037] The second plasmid, pILL834, carried a UreI gene in which all butthe first 21 codons were deleted and replaced with a non-polar cassettecomposed of the aphA-3 kanamycin (Km) resistance gene (25), which hasbeen deleted from its own promoter and terminator regions (19). InShigella flexneri (19) and other organisms (such as Yersiniaenterocolitica, 2) this cassette has been shown not to affect thetranscription of the genes downstream within an operon as long as thesedistal genes have intact translation signals. There is only one basepair separating ureI from ureE (FIG. 1) and ureE does not have an RBS(ribosome binding site) of its own, so the expression of ureI and ureEis transcriptionally and translationally coupled. Therefore, a ureIdeletion was accompanied by the addition of an RBS immediately upstreamfrom ureE. Three intermediates, pILL824, pILL825 and pILL833 (FIG. 2A),were constructed in order to produce the final plasmid, pILL834 (FIG.2A). A 1.8 Kb HpaI-HindIII restriction fragment from pILL753 (9) wasinserted between the EcoRV and HindIII sites of pB3922, to give pILL824.insertion of the H19 adaptor (carrying an RBS and ATG in frame withureE, Table I) into a BclI site overlapping the two first codons of ureEin pILL824 produced pILL825 (FIG. 2A). The BamHI fragment of pILL825 wasthen replaced by a 1.3 Kb blunt-ended PvuII-BamHI fragment from pILL753.This resulted in the reconstitution of a complete ureI gene, and thisplasmid was called pILL833. Finally, pILL834 was obtained by replacementof the HpaI-BglII fragment of pILL833 (thereby deleting all but thefirst 21 codons of ureI) with an 850 bp blunt-ended EcoRI-BamHI fragmentof pUC18K2 containing the non-polar Km cassette (19). TABLE 1 Name andnucleotide sequence of oligonucleotides Oligodeoxynucleotide Primersequence (5′ to 3′) H17 TTTGACTTACTGGGGATCAAGCCTG (SEQ ID NO:1) H19*GATCATTTATTCCTCCAGATCTGGAGGAATAAAT (SEQ ID NO:2) H28GAAGATCTCTAGGACTTGTATTGTTATAT (SEQ ID NO:3) H34 TATCAACGGTGGTATATCCAGTG(SEQ ID NO:4) H35 GCAGTTATTGGTGCCCTTAAACG (SEQ ID NO:5) H50CCGGTGATATTCTCATTTTAGCC (SEQ ID NO:6)  8A GCGAGTATGTAGGTTCAGTA (SEQ IDNO:7)  9B GTGATACTTGAGCAATATCTTCAGC (SEQ ID NO:8) 12BCAAATCCACATAATCCACGCTGAAATC (SEQ ID NO:9)

[0038] Introduction of ureI mutations into H. pylori

[0039]H. pylori ureI mutants were produced by allelic exchange followingelectroporation with a concentrated preparation of pILL823 and pILL834as previously described by Skouloubris et al. (23) from H. pylori strainN6 (12) and from the mouse-adapted H. pylori strain SS1 (Sydney Strain17). Bacteria with chromosomal allelic exchange with pILL883 wereselected on Cm (4 μml) and those with chromosomal allelic exchange withpILL834 on Km (20 μg/ml). It was determined that the desired allelicexchange had taken place in strains N6-823, N6-834. and SS1-834 (FIG. 1)by performing PCR with the appropriate oligonucleotides (Table 1). ThePCR products obtained with genomic DNA of these strains were as expected(i) for strain N6-823: 140 bp with primers H28-H34, 220 bp with H35-9B,and 1.2 Kb with H28-9B, and (ii) for strains N6-834 and SS1-834, 150 bpwith primers H28-H50, 180 bp with H17-12B, and 1 Kb with H28-12B.

[0040] The growth rate of strain N6-834 carrying a non-polar deletion ofureI was compared to that of the parental strain N6. No difference inthe colony size was observed on blood agar medium plates. Identicaldoubling times and stationary phase OD were measured for both strainsgrown in BHI (Oxoid) liquid medium containing 0.2% 3-cyclodextrin(Sigma). Thus, UreI is not essential for H. pylori growth in vitro.

[0041] Urease activity, of H. pylori ureI mutants

[0042] The urease activity of strains N6-823, N6-834. and SS1-834 wasmeasured in vitro as described previously by Cussac et al. (9) andcompared to the activity of the parental strains, N6 and SS 1 (FIG. 1).7Urease activity was almost completely abolished in strain N6-823(0.3±0.1 units). Strains N6-834 and SS l -834, with non-polar ureImutations had wild-type levels of activity (N6-834 and SS1-834: 12-2units; parental strains, N6: 10±1 and SS1: 12±0.4 units).

[0043] The pH optimum of urease produced either from the N6 parentalstrain or from the UreI deficient strain N6-834 was measured andcompared. For both strains, urease has a pH optimum of 8 which isconsistent with the published data.

[0044] These results strongly suggest that the urease-negative phenotypeof the N6-ureI::TnKm-8 (13) and the very weak urease activity of N6-823strains were due to a polar effect of the inserted cassettes on theexpression of the downstream genes ureE and ureF (FIG. 1). Thishypothesis was tested by measuring urease activity of strain N6-823complemented in trans with an E. coli/H. pylori shuttle plasmidexpressing the ureE-F genes. This plasmid, pILL845 (FIG. 2B), wasobtained by insertion of a 2.8 Kb ClaI-BamHI fragment of pILL834(comprising the 3′-end of ureB, the non-polar deletion of ureI andintact ureE and ureF genes) into the corresponding sites of the shuttlevector pHe12 constructed by Heuermann and Haas (15). Strain N6-823 waselectroporated with a DNA preparation of pILL845 as described bySkouloubris et al. ( 23), and transformants were selected on kanamycin(20 μg/ml) and chloramphenicol (4 μg/ml). in strain N6-823 harboringpILL845, wild type urease activity was recovered confirming that thevery low urease activity of strain N6-823 was due to a polar effect onthe expression of the accessory genes ureE-F. In Klebsiella aerogenes,the absence of UreE has little effect on urease activity (4). Incontrast, UreF, as part of the accessory protein complex (UreDFG), isabsolutely required for the production of active urease (21). Thus, byanalogy, it is likely that the phenotype of the H. pylori polar ureImutants was due to the absence of ureF expression.

[0045] The urease structural subunits, UreA and UreB, produced by strainN6 or strain N6-834 were compared with the Western blot technique usinga mixture of antisera directed against each urease subunit. It wasobserved that the amount of each subunit produced by the two strains isidentical. The possibility that urease cellular localization could beaffected in the absence of UreI was examined after cellularfractionation (separating the soluble from the membrane associatedproteins and from the supernatant) of strains N6 and N6-834. Theseexperiments revealed no difference between the urease cellularlocalization in the wild type strain or in the UreI-deficient mutant.These results demonstrate that, at neutral pH, UreI is neitherimplicated in the stabilization of the urease structural subunits nor ina targeting process of urease to a specific cellular compartment.

[0046] Colonization test for the H. pylori SS1-834 mutant in the mouseanimal model

[0047] The mouse model for infection by the H. pylori SS1 strain (SydneyStrain, 17), validated by Chevalier et al. (7) and Ferrero et al. (14),was used to test the function of UreI in vivo. Mice were infected withthe non-polar ureI mutant, SS 1-834, and with the parental strain, SS1,(which had gone through an equivalent number of in vitro subcultures) asa positive control. This experiment was repeated three times andproduced identical results (30). Two independently constructed SS-834mutants were used. The first mutant strain had gone through 30 in vitrosubcultures, the second only 20. Under the same experimental conditions,strain SS1 can undergo more than 80 in vitro subcultures without losingits colonization capacity.

[0048] In each experiment, aliquots (100 μl) containing 10⁶ H. pyloristrain SS1 or SS1-834 bacteria prepared in peptone broth wereadministered orogastrically to 10 mice each (six to eight-weeks oldSwiss specific-pathogen-free mice) as described by Ferrero et al. (I14).Mice were killed four weeks after inoculation. The presence of H. pyloriwas tested with a direct urease test on biopsies performed on half thestomach (14). The remaining gastric tissues were used for quantitativeculture of H. pylori as described by Ferrero et al. (14). In eachexperiment, the stomachs of the ten SS1-infected mice all testedpositive for urease. The bacterial load was between 5×10⁴ and 5×10⁵colony forming units (CFU) per g of stomach. None of the stomachs of themice infected with strain SS1-834 tested positive for urease and no H.pylori cells were cultured from them. Thus, the UreI protein isessential for the H. pylori in vivo survival and/or colonization of themouse stomach.

[0049] UreI is essential for H. pylori resistance to acidity

[0050] Survival to acidic conditions in the presence or absence of 10 mMurea was tested with strains N6 and N6-834. The experimental proceduresdetailed in Skouloubris et al. (30) were based on those described inClyne et al. (8). Exponentially grown bacteria were harvested, washed inPBS (phosphate buffer saline), and approximately 2×10⁸ CFU/ml wereresuspended in PBS of pH 2.2 or pH 7 in the presence or the absence of10 mM urea and incubated at 37EC. After one hour incubation (i)quantitative cultures of the H. pylori strains were performed toevaluate bacterial survival, and (ii) the bacteria were centrifuged andthe pH of the medium was measured. The results obtained are presented inTable 2. In the absence of urea, both strains N6 and N6-834 presentedidentical phenotype, i.e., they were killed at pH 2.2, and survived atpH 7 without modifying the final pH of the medium (Table 2). Afterincubation at pH 7 in the presence of urea, both strains were killedbecause the final pH rose to pH 9. At pH 2.2 in the presence of urea,the parental strain survived well since it was able to raise the pH toneutrality. In contrast, a completely different phenotype was obtainedwith the UreI-deficient strain N6-834 which was unable to raise the pHand whose viability was seriously affected (Table 2).

[0051] Complementation of the UreI-deficient strain N6834 with plasmidpILL850

[0052] Direct implication of the UreI protein in the H. pylori capacityto resist to acidity has been confirmed by trans-complementation withplasmid pILL850 (FIG. 2B restriction map and details of construction).This plasmid [CNCM I-2245 filed on Jun. 28,1999] is derived from the H.pylori/E. coli shuttle vector pHe12 (15). Plasmid pILL850 carries theureI gene under the control of its own promoter and was constructed asfollows: a 1.2 kb BclI restriction fragment of plasmid pILL753 (9) wasintroduced between the BamHI and BclI restriction sites of pHe12 (FIG.2B). Strains N6 and N6-834 were transformed by this plasmid and thephenotype of the complemented strains in the acidity sensitivity testexperiments described above was examined. As shown in Table 2, thephenotype of strain N6-834 complemented by pILL850 is identical to thatof the parental strain N6. Interestingly, the urease activity of thecomplemented strains (measured on sonicated extracts as described inSkouloubris et al. (30)) has been found to be significantly higher ascompared to that of the corresponding strains without pILL850. For thepurpose of the deposit at the CNCM pILL850 is placed into an E. colistrain, MC1061 (Wertman K. F. et al, 1986, Gene 49: 253-262).

[0053] Measurements of ammonium production

[0054] The amount of ammonium produced in the extracellular medium of H.pylori whole cells was measured by an enzymatic assay commercialized bySigma following the supplier's instructions. These experiments wereperformed after incubation of the cells in PBS at different pH valuesand after different incubation times. Such experiments gave an accurateevaluation of ammonium production and excretion in different strains aswell as a measure of the kinetics of this reaction. A control experimentshowed that ammonium production was very low (10-20 μM) in the absenceof urea.

[0055]FIG. 4 depicts the kinetics (0, 3, 5, and 30 min. incubation time)of extracellular ammonium released by the N6 parental strain (panel A)and the UreI-deficient strain N6-834 (panel B) incubated in PBS at pH2.2, pH 5, or pH 7 in the presence of 10 mM urea. The results obtainedindicate that (i) ammonium is largely produced and rapidly released inthe extracellular medium; and (ii) in the N6 wild type strain (FIG. 4,panel A and Table 3) ammonium production is significantly enhanced whenthe extracellular pH is acidic. This effect is already visible at pH 5and is even stronger at pH 2.2. This last observation is consistent withthe results of Scott et al. (31) who suggested urease activation at lowpH. In our experiments, the rapidity of the response to acidity arguesagainst urease activation depending on transcriptional regulation or onde novo protein synthesis.

[0056] Ammonium production was then measured in the UreI-deficientstrain N6-834 (FIG. 4, panel B and Table 3). At neutral pH, kinetics ofammonium production were similar to those of the wild type strain. Incontrast, at pH 5 ammonium production was reduced and delayed ascompared to the wild type strain. A dramatic effect of the absence ofUreI was observed at pH 2.2, where the amount of ammonium was very low,which is consistent with our results showing that UreI is necessary foradaptation to acidity.

[0057] Our results demonstrate that UreI is essential for the resistanceof H. pylori to acidity. In the absence of UreI, urease, althoughpresent in huge amounts, is not able to protect the bacteria against theaggression of acidity. This is consistent with the essential role ofUreI in vivo. During its passage in the acidic stomach lumen, theviability of the UreI-deficient strain is affected. As a consequence,the bacterial load becomes too low to permit colonization. The differentroles proposed for UreI are presented in the “detailed description”section.

[0058] Alignment of the UreI and AmiS protein sequences and twodimensional structure prediction

[0059] A systematic search for UreI homologs in the protein data bankswas carried out. It was determined that H. pylori is not the onlyureolytic bacterium with a ureI gene. Two phylogenetically relatedGram-positive organisms, Streptococcus salivarius, a dental plaquebacterium (6), and Lactobacillus fermentum, a lactic acid bacterium(16), carry genes for UreI-homologs (FIG. 3) located immediatelyupstream from the urease structural genes. The ureI gene has beendetected in various Helicobacter species; the H. felis ureI gene hasbeen entirely sequenced (FIG. 3 and allowed U.S. patent application Ser.No. 08/467,822, the entire contents of which are incorporated herein byreference). PCR experiments have suggested that there is a ureI gene inH. heilmannii (24) and in H. mustelae.

[0060] Sequence similarities between the UreI protein of H. pylori andthe AmiS proteins expressed by the aliphatic amidase operons from P.aeruginosa (27) and Rhodococcus sp. R312 (5) have been reported. InMycobacterium smegmatis, there is an additional AmiS-homolog encoded bya gene, ORF P3, located immediately upstream from an amidase gene (18).

[0061] Alignment of these UreI/AmiS proteins [using the Clustal W(1.60)program] defined strongly conserved stretches of amino acids (FIG. 3).AU but one of these conserved blocks are in highly hydrophobic segments.These regions, each 17 to 22 residues long, are probably folded intotransmembrane ∀-helices (FIG. 3). Six transmembrane regions werepredicted for the proteins from H. pylori, H. felis, and P. aeruginosaand seven for those from Rhodococcus sp. R312 and M. smegmatis (highlyreliable predictions, performed with pHD, a profile fed neural networksystem as described by Rost et al. (22)). The orientation of theUreI/AmiS proteins in the membrane was deduced from the charges of theintercalated hydrophilic regions, which are short in these proteins(FIG. 3). The first five such regions are poorly conserved and ofvarious length. The last interhelical segment common to these proteinsis significantly more conserved than the others. This region predictedto be intracellular may be the active site of UreI or a site ofmultimerization or interaction with an intracellular partner. Theseresults strongly suggest that the members of the UreI/AmiS family, foundin both Gram-positive and -negative bacteria, are integral membraneproteins. These proteins have no signal sequence and should therefore beinserted into the cytoplasmic membrane in Gram-negative bacteria.

[0062] Two peptides, selected from the UreI sequence, were synthesizedand injected into two rabbits to obtain serum containing polyclonalantibodies directed against UreI. One peptide corresponds to the firstpredicted intracellular loop of UreI (from residue nB 15 to 31, see FIG.3) and the second one to the second predicted extracellular loop of UreI(from residue nB 118 to 134, see FIG. 3. These sera are presently beingtested and if proven to recognize the UreI protein will allow us toprecisely define the localization of this protein and to verify thepredicted UreI two-dimensional structure presented in FIG. 3.

[0063] The references cited herein are specifically incorporated byreference in their entirety. TABLE 2 Effect of the presence of urea atpH 7, 5 or 2.2 on (i) the viability of different H. pylori strains and(ii) the extracellular pH (indicated as final pH). The experimentalprocedures are described in reference 30 and in the examples. Strain N6is the parental strain and strain N6-834 the Urel-deficient mutant.Plasmid pILL850 is derived from a E. coli/ H. pylori shuttle vector, itcarries the urel gene and complements the urel mutation of strainN6-834. urea strains initial pH final pH 10 mM H. pylori CFU/ml N6 2.22.26 − 0 N6 2.2 6.6 +   8 × 10⁷ N6 7 6.98 −   2 × 10⁸ N6 7 8.88 + 0N6-834 2.2 2.2 − 0 N6-834 2.2 2.37 +   7 × 10⁵ N6-834 7 7.1 − 3.5 × 10⁷N6-834 7 9.05 + 0 N6-834 + pILL850 2.2 2.3 − 0 N6-834 + pILL850 2.26.9 + 1.3 × 10⁸ N6-834 + pILL850 7 7.1 − 1.7 × 10⁸ N6-834 + pILL850 79 + 0

[0064] TABLE 3 medium [NH4] Strain pH minutes mM N6 7,0 0 3.5 N6 7,0 34.4 N6 7,0 5 3.1 N6 7,0 30 5.6 N6 5,0 0 12.8 N6 5,0 3 9.3 N6 5,0 5 11.8N6 5,0 30 16,0 N6 2,2 0 6.7 N6 2,2 3 9,0 N6 2,2 5 11,0 N6 2,2 30 20,0N6-834 7,0 0 2,.7 N6-834 7,0 3 2.8 N6-834 7,0 5 3.8 N6-834 7,0 30 5.8N6-834 5,0 0 1.4 N6-834 5,0 3 1.7 N6-834 5,0 5 2.9 N6-834 5,0 30 4.6N6-834 2,2 0 0.9 N6-834 2,2 3 0.6 N6-834 2,2 5 0.7 N6-834 2,2 30 1.3

REFERENCES

[0065] 1. Akada, J. K., M. Shirai, H. Takeuchi, M. Tsuda, and T.Nakazawa. 1997. Transcriptional analysis of urease structural gene andthe ureI gene in Helicobacter pylori. Gut. 41:A9.

[0066] 2. Allaoui, A., Schulte, R. and G. R. Cornelis. 1995. Mutationalanalysis of the Yersinia enterocolitica virC operon: characterization ofyscE, F, G, H, I, J, K required for Yop secretion and ysch encodingYopR. Mol. Microbiol. 18:343-355.

[0067] 3. Bauerfeind, P., R. M. Garner, and H. L. T. Mobley. 1996.Allelic exchange mutagenesis of mxA in Helicobacter pylori results inreduced nickel transport and urease activity. Infect. Immun.64:2877-2880.

[0068] 4. Brayman, T. G., and R. T. Hausinger. 1996. Purification,characterization, and functional anal%sis of a truncated Klebsiellaaerogenes UreE urease accessory protein lacking the Histidine-Richcarboxyl terminus. J. Bacteriol. 178:5410-5416.

[0069] 5. Chebrou, H., F. Bigey, A. Arnaud, and P. Galzy. 1996. Amidemetabolism: a putative ABC transporter in Rhodococcus sp. R312. Gene.182:215-218.

[0070] 6. Chen, Y.-Y. M., K. A. Clancy, and R. A. Burne. 1996.Streptococcus salivarius urease: genetic and biochemicalcharacterization and expression in a dental plaque Streptococcus.Infect. Immun. 64:585-592.

[0071] 7. Chevalier, C., J.-M. Thiberge, R. L. Ferrero, and A. Labigne.1999. Essential role of Helicobacter pylori g-Glutamyltranspeptidase(GGT) for the colonization of the gastric mucosa in mice. Mol.Microbiol.31:1359-1372.

[0072] 8. Clyne, M., A. Labigne, and B. Drumm. 1995. Helicobacter pylorirequires an acidic environment to survive in the presence of urea.Infect. Immun. 63:1669-1673.

[0073] 9. Cussac, V., R. L. Ferrero, and A. Labigne. 1992. Expression ofHelicobacter pylori urease genes in Escherichia coli grown undernitrogen-limiting conditions. J. Bacteriol. 174:2466-2473.

[0074] 10. Dunn. B. E., H. Cohen, and M. Blaser. 1997. Helicobacterpylori. Clin. Microbiol. Rev. 10:720-74 .

[0075] 11. Eaton, K. A., and S. Krakowka. 1994. Effect of gastric pH onurease-dependent colonization of gnotobiotic piglets by Helicobacterpylori. Infect. Immun. 62:3604-3607.

[0076] 12. Ferrero, R. L., V. Cussac, P. Courcoux, and A. Labigne. 1992.Construction of isogenic urease-negative mutants of Helicobacter pyloriby allelic exchange. J. Bacteriol. 174:4212-4217.

[0077] 13. Ferrero, R. L., V. Cussac, P. Courcoux, and A. Labigne. 1994.Construction of isogenic mutants of Helicobacter pylori deficient inurease activity. pp 179-182. In Basic and Clinical Aspects of H. pyloriinfection. Springer-Verlag Berlin Heidelberg.

[0078] 14. Ferrero, R. L., J.-M. Thiberge, M. Huerre, and A. Labigne.1998. Immune responses of specific-pathogen-free mice to chronicHelicobacter pylori (strain SS1) infection. Infect. Immun. 66:1349-1355.

[0079] 15. Heuermann, D., and R. Haas. 1998. A stable shuttle vectorsystem for efficient genetic complementation of Helicobacter pyloristrains by complementation and conjugation. Mol. Gen. Genet.257:519-528.

[0080] 16. Kakimoto, S., Y. Sumino, K. Kawahara, E. Yamazaki, and I.Nakatsui. 1990. Purification and characterization of acid urease fromLactobacillus fermentum. Appl. Microbiol. & Biotechnol. 32:538-543.

[0081] 17. Lee, A., J. O'Rourke, M. Corazon De Ungria, B. Robertson, G.Daskalopoulos, and M. F. Dixon. 1997. A standardized mouse model ofHelicobacter pylori infection: introducing the Sydney Strain.Gastroenterology. 112:1386-1397.

[0082] 18. Mahenthiralingam, E., P. Draper, E. O. Davis, and M. J.Colston. 1993. Cloning and sequencing of the gene which encodes thehighly inducible acetamidase of Mycobacterium smegmatis. J. Gen.Microbiol. 139:575-583.

[0083] 19. Menard, R., P. J. Sansonetti, and C. Parsot. 1993. Nonpolarmutagenesis of the ipa genes define; IpaB, IpaC, and IpaD as effectorsof Shigella flexneri entry into epithelial cells. J. Bacteriol.175:5899-5906.

[0084] 20. Mobley, H. L. T., M. D. Island, and R. P. Hausinger. 1995.Molecular biology of ureases. Microbiol. Rev. 59:451-480.

[0085] 21. Moncrief, M. B. C., and R. P. Hausinger. 1997.Characterization of UreG, identification of a UreD-UreF-UreG complex,and evidence suggesting that a nucleotide-binding site in UreG isrequired for in vivo metallocenter assembly of Klebsiella aerogenesurease. J. Bacteriol. 179:4081-4086.

[0086] 22. Rost, B., R. Casadio, P. Fariselli, and C. Sander. 1995.Prediction of helical transmembrane segments at 95% accuracy. Prot.Science. 4:521-533.

[0087] 23. Skouloubris, S., A. Labigne, and H. DeReuse. 1997.Identification and characterization of an aliphatic amidase inHelicobacter pylori. Mol. Microbiol. 25:989-998.

[0088] 24. Solnick, J. V., J. O'Rourke, A. Lee, and L. S. Tompkins.1994. Molecular analysis of urease genes from a newly identifieduncultured species of Helicobacter. Infect. Immun. 62:163,-1638.

[0089] 25. Trieu-Cuot, P., G. Gerbaud, T. Lambert and P. Courvalin,1985. In vivo transfer of genetic information between Gram-positive andGram-negative bacteria. EMBO J. 4:3583-3587.

[0090] 26. Williams, C. L., T. Preston, M. Hossack, C. Slater, and K. E.L. McColl. 1996. Helicobacter pylori utilizes urea for amino acidsynthesis. FEMS Immunol. Med. Microbiol. 13:87-94.

[0091] 27. Wilson, S. A., R. J. Williams, L. H. Pearl, and R. E. Drew,.1995. Identification of two new genes in the Pseudomonas aeruginosaamidase operon, encoding an ATPase (AmiB) and a putative integralmembrane protein (AmiS). J. Biol. Chem. 270:18818-18824.

[0092] 28. Mendz, G. L. and S. L. Mazell. 1996. The Urea Cycle ofHelicobacter pylori. Microbiology 142:2959-2967.

[0093] 29. Nicholson, E. B., E. A. Concaugh and H. L. T. Mobley. 1991.Proteus mirabilis urease: use of ureA-lacZ fusion demonstrates thatinduction is highly specific for urea. Infection and Immunity.59(10):3360-3365.

[0094] 30. Skouloubris, S., J.-M. Thiberge, A. Labigne and H. De Reuse(1998) The Helicobacter pylori UreI protein is not involved in ureaseactivity but is essential for bacterial survival in vivo. Infect. Immun.66: 4517-74521.

[0095] 31. Scott, D. R., D. Weeks, C. Hong, S. Postius, K. Melchers andG. Sachs (1998) The role of internal urease in acid resistance ofHelicobacter pylori. Gastroenterology. 114: 58-70.

1 18 1 25 DNA Artificial Sequence Description of Artificial SequencePrimer 1 tttgacttac tggggatcaa gcctg 25 2 34 DNA Artificial SequenceDescription of Artificial Sequence Adaptor sequence 2 gatcatttattcctccagat ctggaggaat aaat 34 3 29 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 3 gaagatctct aggacttgta ttgttatat 29 4 23DNA Artificial Sequence Description of Artificial Sequence Primer 4tatcaacggt ggtatatcca gtg 23 5 23 DNA Artificial Sequence Description ofArtificial Sequence Primer 5 gcagttattg gtgcccttaa acg 23 6 23 DNAArtificial Sequence Description of Artificial Sequence Primer 6ccggtgatat tctcatttta gcc 23 7 20 DNA Artificial Sequence Description ofArtificial Sequence Primer 7 gcgagtatgt aggttcagta 20 8 25 DNAArtificial Sequence Description of Artificial Sequence Primer 8gtgatacttg agcaatatct tcagc 25 9 27 DNA Artificial Sequence Descriptionof Artificial Sequence Primer 9 caaatccaca taatccacgc tgaaatc 27 10 195PRT Helicobacter pylori 10 Met Leu Gly Leu Val Leu Leu Tyr Val Gly IleVal Leu Ile Ser Asn 1 5 10 15 Gly Ile Cys Gly Leu Thr Lys Val Asp ProLys Ser Thr Ala Val Met 20 25 30 Asn Phe Phe Val Gly Gly Leu Ser Ile IleCys Asn Val Val Val Ile 35 40 45 Thr Tyr Ser Ala Leu Asn Pro Thr Ala ProVal Glu Gly Ala Glu Asp 50 55 60 Ile Ala Gln Val Ser His His Leu Thr AsnPhe Tyr Gly Pro Ala Thr 65 70 75 80 Gly Leu Leu Phe Gly Phe Thr Tyr LeuTyr Ala Ala Ile Asn His Thr 85 90 95 Phe Gly Leu Asp Trp Arg Pro Tyr SerTrp Tyr Ser Leu Phe Val Ala 100 105 110 Ile Asn Thr Ile Pro Ala Ala IleLeu Ser His Tyr Ser Asp Met Leu 115 120 125 Asp Asp His Lys Val Leu GlyIle Thr Glu Gly Asp Trp Trp Ala Ile 130 135 140 Ile Trp Leu Ala Trp GlyVal Leu Trp Leu Thr Ala Phe Ile Glu Asn 145 150 155 160 Ile Leu Lys IlePro Leu Gly Lys Phe Thr Pro Trp Leu Ala Ile Ile 165 170 175 Glu Gly IleLeu Thr Ala Trp Ile Pro Ala Trp Leu Leu Phe Ile Gln 180 185 190 His TrpVal 195 11 196 PRT Helicobacter felis 11 Met Leu Gly Leu Val Leu Leu TyrVal Ala Val Val Leu Ile Ser Asn 1 5 10 15 Gly Val Ser Gly Leu Ala AsnVal Asp Ala Lys Ser Lys Ala Ile Met 20 25 30 Asn Tyr Phe Val Gly Gly AspSer Pro Leu Cys Val Met Trp Ser Leu 35 40 45 Ser Ser Tyr Ser Thr Phe HisPro Thr Pro Pro Ala Thr Gly Pro Glu 50 55 60 Asp Val Ala Gln Val Ser GlnHis Leu Ile Asn Phe Tyr Gly Pro Ala 65 70 75 80 Thr Gly Leu Leu Phe GlyPhe Thr Tyr Leu Tyr Ala Ala Ile Asn Asn 85 90 95 Thr Phe Asn Leu Asp TrpLys Pro Tyr Gly Trp Tyr Cys Leu Phe Val 100 105 110 Thr Ile Asn Thr IlePro Ala Ala Ile Leu Ser His Tyr Ser Asp Ala 115 120 125 Leu Asp Asp HisArg Leu Leu Gly Ile Thr Glu Gly Asp Trp Trp Ala 130 135 140 Phe Ile TrpLeu Ala Trp Gly Val Leu Trp Leu Thr Gly Trp Ile Glu 145 150 155 160 CysAla Leu Gly Lys Ser Leu Gly Lys Phe Val Pro Trp Leu Ala Ile 165 170 175Val Glu Gly Val Ile Thr Ala Trp Ile Pro Ala Trp Leu Leu Phe Ile 180 185190 Gln His Trp Ser 195 12 46 PRT Lactobacillus fermentum 12 Ile Leu TrpLeu Thr Gly Phe Leu Thr Asn Asn Leu Lys Met Asn Leu 1 5 10 15 Gly LysPhe Pro Gly Tyr Leu Gly Ile Ile Glu Gly Ile Cys Thr Ala 20 25 30 Trp IlePro Gly Phe Leu Met Leu Leu Asn Tyr Trp Pro Asn 35 40 45 13 129 PRTStreptococcus salivarius 13 Ile Leu Asn Ile Ile Val Ile Ala Tyr Gly AlaCys Thr Gly Gln Gly 1 5 10 15 Ala Glu Trp Phe Tyr Gly Ser Ala Thr GlyLeu Leu Phe Ala Phe Thr 20 25 30 Tyr Leu Tyr Ser Ala Ile Asn Thr Ile PheAsp Phe Asp Gln Arg Leu 35 40 45 Tyr Gly Trp Phe Ser Leu Phe Val Ala IleAsn Thr Leu Pro Ala Gly 50 55 60 Ile Leu Cys Leu Thr Ser Gly Tyr Gly GlyAsn Ala Trp Tyr Gly Ile 65 70 75 80 Ile Trp Phe Leu Trp Gly Ile Leu TrpLeu Thr Ala Phe Ile Glu Ile 85 90 95 Asn Leu Lys Lys Asn Leu Gly Lys PheVal Pro Tyr Leu Ala Ile Phe 100 105 110 Glu Gly Ile Val Thr Ala Trp IlePro Gly Leu Leu Met Leu Trp Gly 115 120 125 Lys 14 213 PRT Mycobacteriumsmegmatis 14 Met Gly Gly Val Gly Leu Phe Tyr Val Gly Ala Val Leu Ile IleAsp 1 5 10 15 Gly Leu Met Leu Leu Gly Arg Ile Ser Pro Arg Gly Ala ThrPro Leu 20 25 30 Asn Phe Phe Val Gly Gly Leu Gln Val Val Thr Pro Thr ValLeu Ile 35 40 45 Leu Gln Ser Gly Gly Asp Ala Ala Val Ile Phe Ala Ala SerGly Leu 50 55 60 Tyr Leu Phe Gly Phe Thr Tyr Leu Trp Val Ala Ile Asn AsnVal Thr 65 70 75 80 Asp Trp Asp Gly Glu Gly Leu Gly Trp Phe Ser Leu PheVal Ala Ile 85 90 95 Ala Ala Leu Gly Tyr Ser Trp His Ala Phe Thr Ala GluAla Asp Pro 100 105 110 Ala Phe Gly Val Ile Trp Leu Leu Trp Ala Val LeuTrp Phe Met Leu 115 120 125 Phe Leu Leu Leu Gly Leu Gly His Asp Ala LeuGly Pro Ala Val Gly 130 135 140 Phe Val Ala Val Ala Glu Gly Val Ile ThrAla Ala Val Pro Ala Phe 145 150 155 160 Leu Ile Val Ser Gly Asn Trp GluThr Gly Pro Leu Pro Ala Ala Val 165 170 175 Ile Ala Val Ile Gly Phe AlaAla Val Val Leu Ala Tyr Pro Ile Gly 180 185 190 Arg Arg Leu Ala Ala ProSer Val Thr Asn Pro Pro Pro Ala Ala Leu 195 200 205 Ala Ala Thr Thr Arg210 15 206 PRT Rhodococcus sp. 15 Met Gly Ser Val Gly Leu Leu Tyr ValGly Ala Val Leu Phe Val Asn 1 5 10 15 Gly Leu Met Leu Leu Gly Thr ValPro Val Arg Ser Ala Ser Val Leu 20 25 30 Asn Leu Phe Val Gly Ala Leu GlnCys Val Val Pro Thr Val Met Leu 35 40 45 Ile Gln Ala Gln Gly Asp Ser SerAla Val Leu Ala Ala Ser Gly Leu 50 55 60 Tyr Leu Phe Gly Phe Thr Tyr LeuTyr Val Gly Ile Ser Asn Leu Ala 65 70 75 80 Gly Phe Glu Pro Glu Gly IleGly Trp Phe Ser Leu Phe Val Ala Cys 85 90 95 Ala Ala Leu Val Tyr Ser PheLeu Ser Phe Thr Val Ser Asn Asp Pro 100 105 110 Val Phe Gly Val Ile TrpLeu Ala Trp Ala Ala Leu Trp Thr Leu Phe 115 120 125 Phe Leu Val Leu GlyLeu Gly Arg Glu Asn Leu Ser Arg Phe Thr Gly 130 135 140 Trp Ala Ala IleLeu Leu Ser Gln Pro Thr Cys Thr Val Pro Ala Phe 145 150 155 160 Leu IleLeu Thr Gly Asn Phe His Thr Thr Pro Ala Val Ala Ala Gly 165 170 175 TrpAla Gly Ala Leu Leu Val Leu Leu Gly Leu Ala Lys Ile Leu Ala 180 185 190Ala Pro Lys Ala Ala Val Pro Gln Pro Arg Pro Val Phe Asn 195 200 205 16171 PRT Pseudomonas aeruginosa 16 Met Leu Gly Leu Val Leu Leu Tyr ValGly Ala Val Leu Phe Leu Asn 1 5 10 15 Ala Val Trp Leu Leu Gly Lys IleSer Gly Arg Glu Val Ala Val Ile 20 25 30 Asn Phe Leu Val Gly Val Leu SerAla Cys Val Ala Phe Tyr Leu Ile 35 40 45 Phe Ser Ala Ala Ala Gly Gln GlySer Leu Lys Ala Gly Ala Leu Thr 50 55 60 Leu Leu Phe Ala Phe Thr Tyr LeuTrp Val Ala Ala Asn Gln Phe Leu 65 70 75 80 Glu Val Asp Gly Lys Gly LeuGly Trp Phe Cys Leu Phe Val Ser Leu 85 90 95 Thr Ala Cys Thr Val Ala IleGlu Ser Phe Ala Gly Ala Ser Gly Pro 100 105 110 Phe Gly Leu Trp Asn AlaVal Asn Trp Thr Val Trp Ala Leu Leu Trp 115 120 125 Phe Cys Phe Phe LeuLeu Leu Gly Leu Ser Arg Gly Ile Gln Lys Pro 130 135 140 Val Ala Tyr LeuThr Leu Ala Ser Ala Ile Phe Thr Ala Trp Leu Pro 145 150 155 160 Gly LeuLeu Leu Leu Gly Gln Val Leu Lys Ala 165 170 17 19 DNA ArtificialSequence Description of Artificial Sequence Primer 17 tgggtgtgagatgatcata 19 18 53 DNA Artificial Sequence Description of ArtificialSequence Adaptor sequence 18 tgggtgtgag atgatcattt attcctccag atctggaggaataaatgatc ata 53

We claim:
 1. An in vitro method for identifying a molecule capable ofinhibiting the growth or survival of Helicobacter in vivo, comprisingthe steps of: (a) contacting a parental Helicobacter with said moleculein a biological sample, (b) testing and comparing the response toextracellular pH and the sensitivity to acidity of the parental strainto a strain deficient in UreI and/or of a UreI deficient straincomplemented with a plasmid carrying ureI in the presence or absence ofsaid active molecule: and (c) selecting said molecule displaying adifferential effect on the parental strain as compared to the UreIdeficient strain.
 2. The method according to claim 1, wherein the degreeof acidity sensitivity is measured in step (b).
 3. The method accordingto claim 1, wherein the molecules are specific to UreI.
 4. The methodaccording to claim 1, wherein the Helicobacter strain is selected fromthe group consisting of Helicobacter pylori, helicobacter felis,Helicobacter heilmannii, Helicobacter mustelae, Helicobacter canis,Helicobacter bilis, Helicobacter hepaticus, Helicobacter muridarum, andHelicobacter troguntum.
 5. A molecule capable of inhibiting the growthor survival of H. pylori in vivo identified by the method according toclaim
 1. 6. A method of treating or preventing H. pylori infection inhumans or animals comprising the step of administering a moleculeaccording to claim 5 together with a pharmaceutically acceptable carrierto a human or animal in need of such treatment.
 7. A method ofpreventing or treating H. pylori infection comprising the step ofadministering a molecule capable of inhibiting the growth or survival ofH. pylori in vivo to a human or animal in need of such treatment.
 8. Themethod according to claim 7, wherein the molecule is transported insidethe H. pylori cell due to a high affinity for UreI.
 9. The methodaccording to claim 7, wherein the molecule inactivates UreI byinhibiting its properties in H. pylori resistance to acidity.
 10. Themethod according to claim 7, wherein the molecule inactivates UreI byinhibiting its properties as a transporter.
 11. The method according toclaim 7, wherein the molecule inactivates UreI by inhibiting aninteraction, between UreI and other H. pylori proteins.
 12. The methodaccording to claim 7, wherein the molecule is capable of intracellularinhibition of urease in H. pylori.
 13. A molecule capable of inhibitingthe growth or survival of Helicobacter pylori in vivo said moleculebeing capable of interacting directly or indirectly with UreI protein orUreI activity or with the corresponding ureI gene.
 14. A moleculeaccording to claim 13, wherein the molecule is transported inside the H.pylori cell due to a high affinity for ureI.
 15. A molecule according toclaims 9 and 13, wherein the molecule inactivates UreI protein bydirectly binding to ureI.
 16. A molecule according to claims 10 and 13,wherein the molecule is specifically inhibiting UreI transporterproperties either in ammonia export or in urea transport (export orimport).
 17. A molecule according to claims 11 and 13 wherein themolecule is capable of specifically inhibiting an interaction betweenUreI and other H. pylori proteins.
 18. A molecule according to claims 12and 13 wherein the molecule is capable of intracellular inhibition of H.pylori urease.
 19. An immunogenic composition comprising all or part ofH. pylori UreI and a pharmaceutically acceptable carrier.
 20. A methodof preventing Helicobacter pylori infection comprising the step ofadministering an immunogenic composition according to claim
 19. 21.Monoclonal or polyclonal antibodies that specifically recognize all orpart of H. pylori UreI.
 22. A recombinant strain of H. pylori comprisinga modified ureI gene, wherein the product of the gene contributes to theattenuation of the bacteria's ability to survive in vivo.
 23. Arecombinant strain according to claim 22 named H. pylori N6-834 mutantdeposited at C.N.C.M. on Jun. 28,
 1999. 24. A plasmid pILL 850,deposited at the C.N.C.M. on Jun. 28, 1999 under Accession Number1-2245, able to stably replicate in H. pylori, carrying the ureI geneand complementing the H. pyroli N6-834 mutant.